Hydroxyurea (HU) is effectively used in the management of &#946;-hemoglobinopathies by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear. We previously reported that overexpression of the HU-induced SAR1 gene closely mimics the known effects of HU on K562 and CD34(+) cells, including &#947;-globin induction and cell-cycle regulation. Here, we show that HU stimulated nuclear factor-&#954;B interaction with its cognate-binding site on the SAR1 promoter to regulate transcriptional expression of SAR1 in K562 and CD34(+) cells. Silencing SAR1 expression not only significantly lowered both basal and HU-elicited HbF production in K562 and CD34(+) cells, but also significantly reduced HU-mediated S-phase cell-cycle arrest and apoptosis in K562 cells. Inhibition of c-Jun N-terminal kinase (JNK)/Jun phosphorylation and silencing of Gi&#945; expression in SAR1-transfected K562 and CD34(+) cells reduced both &#947;-globin expression and HbF level, indicating that activation of Gi&#945;/JNK/Jun proteins is required for SAR1-mediated HbF induction. Furthermore, reciprocal coimmunoprecipitation assays revealed an association between forcibly expressed SAR1 and Gi&#945;2 or Gi&#945;3 proteins in both K562 and nonerythroid cells. These results indicate that HU induces SAR1, which in turn activates &#947;-globin expression, predominantly through the Gi&#945;/JNK/Jun pathway. Our findings identify SAR1 as an alternative therapeutic target for &#946;-globin disorders.